Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane.
The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.
Pavlica
R J
RJ
Department of Biological Sciences, St. John's University, Jamaica, NY 11439.
Hesler
C B
CB
Lipfert
L
L
Hirshfield
I N
IN
Haldar
D
D
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008930:Mitochondria, Liver / Q000032:analysis*; D008933:Mitochondrial Swelling; D010455:Peptides / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D008565:Membrane Proteins; D010455:Peptides